The bases of DNA are continually damaged by environmental toxicants and reactive cellular metabolites. The repair of this damage is critical for the maintenance of our genomic integrity. The DNA glycosylases that initiate base excision repair (BER) locate damaged bases within a vast excess of normal DNA and initiate the removal of the chemically modified base. A growing body of experimental data indicates that DNA glycosylases hand off their abasic DNA product to the enzymes that catalyze subsequent steps of base excision repair. These protein-protein interactions during BER not only dictate the order, timing and progression of this repair pathway but also sequester potentially reactive and toxic reaction intermediates from other DNA metabolizing enzymes. In Project 1 of his proposal, we will exploit the complementary expertise of the Samson, Ellenberger and Tomkinson research groups to explore the molecular mechanisms that coordinate the multiple steps of BER. We will use a combination of biochemical, genetic and structural studies. The contributions of the Expression and Molecular Cell Biology (EMB) and the Structural Cell Biology (SCB) Cores will be critical for the success of this project. Specifically, the EMB Core will develop vectors that express well-folded proteins and/or protein fragments that assemble into complexes amenable to structural analysis. Using the SIBYLS beamline in the SCB Core, the shape and conformation of protein complexes will be revealed by SAXS and the atomic structure of protein complexes will be determined by x-ray crystallography. Recent biochemical and genetic studies have revealed an unexpectedly complex interplay between different cellular DNA repair pathways. Thus the study of different repair pathways within the Structural Biology of DNA Repair (SDBR) program will produce synergistic interactions between investigators with different expertise and enhance our understanding of DNA repair in vivo. In particular, our studies of BER are complementary to those of transcription-coupled BER and replication-associated BER in Project 2. Since PCNA is required for DNA mismatch repair and DNA synthesis associate with DNA double- strand break, our proposed studies on the role of PCNA in BER will also be relevant to Projects 3, 4 and 5.